FURO[3,4-b]PYRAN COMPOUNDS AND PHARMACEUTICAL USES

ABSTRACT

Furo[3,4-b]pyran compounds similar in chemical structure to the natural product known as TAN-2483B and their use for treating cancer, osteoporosis, Type 2 diabetes, or immune diseases.

TECHNICAL FIELD

The invention relates to compounds that are structurally related toknown biologically active natural products. The invention furtherrelates to pharmaceutical compositions containing the compounds and tothe use of the compounds for treating diseases. In particular, theinvention relates to furo[3,4-b]pyran compounds similar in chemicalstructure to the natural product known as TAN-2483B.

BACKGROUND OF THE INVENTION

Chemical compounds that occur in nature have been an enormously valuablesource of potential therapeutic agents for the treatment of manydiseases. Following isolation from their source and purification,naturally occurring compounds may be used as active ingredients inpharmaceutical compositions. However, more commonly, derivatives orstructural analogues of naturally occurring compounds become the activeingredients of pharmaceutical compositions. Although there are manyinstances of successful therapeutic treatments based on naturallyoccurring compounds, there remains a constant ongoing need for new andimproved medicines. The search for natural products that may assisttherefore continues.

Following the discovery of a class of compounds in which at least somemembers have potentially useful biological activity, there is usually astrong interest in the production of selected compounds from the class,initially for further development and ultimately for production on alarge scale for marketing and sales. Production may be possible bysimple isolation from a natural source, but typically this is notpossible and synthetic or semi-synthetic processes are required to makesufficient quantities of the useful compounds.

One class of chemical compounds that has proven to be a rich source ofcompounds having biological activity is the furo[3,4-b]pyrans. Bioactivenatural products incorporating the furo[3,4-b]pyran-5-one bicyclicsystem have been isolated from a variety of fungal sources.Fusidilactones A, B, D and E,^(1,2) massarilactones B and D,³⁻⁵TAN-2483A and TAN-2483B,⁶ and waol A⁷⁻¹¹ all contain this ring system.These fungal secondary metabolites display a variety of bioactivities,ranging from antibacterial to anti-tumour properties.

Syntheses of some members of the furo[3,4-b]pyran family of naturalproducts, namely (−)-TAN-2483A, massarilactone B and waol A, areknown.⁹⁻¹¹ These natural products either incorporate a degree ofunsaturation across the fused 4a-7a bond (e.g. massarilactone B andfusidilactone A) or possess a cis-relationship between H-2 and H-7a(e.g. (−)-TAN-2483A and waol A). In contrast to the other members ofthis family, (−)-TAN-2483B, isolated from a Japanese filamentousfungus,⁶ has a trans-relationship between H-2 and H-7a, and thereforepresents different synthetic challenges.

(−)-TAN-2483A and (−)-TAN-2483B, isolated from fermentation of thefilamentous fungus NR2329 (FERM BP-5905) in a culture medium, exhibitinhibition of c-Src (sarcoma) kinase and PTH-induced bone resorption.Therefore they have potential relevance to human pharmaceutics,including in cancer therapy and osteoporosis prevention or treatment.The synthesis of the furo[3,4-b]pyran core of (−)-TAN-2483B has beenreported.¹² The synthetic route is via a D-mannose-derived cyclopropanekey intermediate. Employing this general synthetic methodology, theinventors have investigated the synthesis of analogues of (−)-TAN-2483Bwith the objective of then determining their biological activities in arange of assays and assessing their potential as pharmaceutical agentsfor treating certain diseases. The inventors have now found thatanalogues of (−)-TAN-2483B show inhibitory activity against a range ofkinase enzymes and also inhibit the growth of certain cancer cell lines.

It is therefore an object of the invention to provide novel compoundsand their use for treating diseases, or to at least provide a usefulalternative to existing pharmaceutical treatments.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a compound of theformula:

wherein

-   -   R¹ and R² may each be H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆        alkenyl, C₂-C₆ alkynyl, aryl, CO₂H, CO₂alkyl, or C(═O)alkyl,        wherein each alkyl, alkoxy, alkenyl, alkynyl or aryl group may        optionally be substituted with OH, NH₂, halogen, alkoxy, acyloxy        or aryl; and    -   R³ is H, C₁-C₆ alkyl, C₁-C₆ acyl, aryl, benzyl or trialkylsilyl;    -   provided that R¹ is not CH₃ when R² and R³ are both H or when R²        is H and R³ is acetyl;

or a pharmaceutically acceptable salt thereof.

In a second aspect of the invention there is provided a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable carrier.

In another aspect of the invention there is provided a method oftreating cancer, osteoporosis, Type 2 diabetes or an immune diseasecomprising administering a pharmaceutically effective amount of acompound of the invention to a patient requiring treatment.

DETAILED DESCRIPTION Definitions

The term “alkyl” means any saturated hydrocarbon radical, and isintended to include both straight- and branched-chain alkyl groups.Examples of alkyl groups include: methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl,1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-ethylpropyl,n-hexyl and 1-methyl-2-ethylpropyl.

The term “alkenyl” means any hydrocarbon radical having at least onedouble bond, and is intended to include both straight- andbranched-chain alkenyl groups. Examples of alkenyl groups include:ethenyl, n-propenyl, iso-propenyl, n-butenyl, iso-butenyl, sec-butenyl,t-butenyl, n-pentenyl, 1,1-dimethylpropenyl, 1,2-dimethylpropenyl,2,2-dimethylpropenyl, 1-ethylpropenyl, 2-ethylpropenyl, n-hexenyl and1-methyl-2-ethylpropenyl.

The term “alkynyl” means any hydrocarbon radical having at least onecarbon-carbon triple bond, and is intended to include both straight- andbranched-chain alkynyl groups. Examples of alkynyl groups include:ethynyl, n-propynyl and n-butynyl.

The term “aryl” means an aromatic radical having 4 to 18 carbon atomsand includes heteroaromatic radicals. Examples include monocyclicgroups, as well as fused groups such as bicyclic groups and tricyclicgroups. Examples include phenyl, indenyl, 1-naphthyl, 2-naphthyl,azulenyl, heptalenyl, biphenyl, indacenyl, acenaphthyl, fluorenyl,phenalenyl, phenanthrenyl, anthracenyl, cyclopentacyclooctenyl, andbenzocyclooctenyl, pyridyl, pyrrolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazolyl, and tetrazolyl.

The term “alkoxy” means an OR group, where R is alkyl as defined above.

The term “acyl” means a —(C═O)R group, where R is alkyl as definedabove.

The term “acyloxy” means a —O(C═O)R group, where R is alkyl as definedabove.

The term “pharmaceutically acceptable salt” is intended to apply tonon-toxic salts derived from inorganic or organic acids, including, forexample, the following acid salts: acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate,glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate,p-toluenesulfonate, salicylate, succinate, sulfate, tartrate,thiocyanate and undecanoate.

The “

” in the compound formula means that the methyl group attached to thefuran ring at the position adjacent to the furan ring oxygen atom maybein either the α- or β-stereochemistry.

Compounds of the Invention and their Uses

The invention relates to compounds of the formula:

wherein

-   -   R¹ and R² may each be H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆        alkenyl, C₂-C₆ alkynyl, aryl, CO₂H, CO₂alkyl, or C(═O)alkyl,        wherein each alkyl, alkoxy, alkenyl, alkynyl or aryl group may        optionally be substituted with OH, NH₂, halogen, alkoxy, acyloxy        or aryl; and    -   R³ is H, C₁-C₆ alkyl, C₁-C₆ acyl, aryl, benzyl or trialkylsilyl;    -   provided that R¹ is not CH₃ when R² and R³ are both H or when R²        is H and R³ is acetyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments of the invention, R¹ and R² may each be H, C₁-C₆alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, CO₂H, CO₂alkyl,or C(═O)alkyl, wherein each alkyl, alkoxy, alkenyl, alkynyl or arylgroup may optionally be substituted with OH, NH₂, halogen, or aryl; andR³ is H, C₁-C₆ alkyl, aryl, benzyl or trialkylsilyl.

In some embodiments of the invention, one of R¹ and R² is C₁-C₆ alkyland the other is H. For example, R¹ may be C₁-C₆ alkyl and R² is H. Inother embodiments, one of R¹ and R² is CH₂OH and the other is H. Thus,R¹ may be H and R² is CH₂OH, or R¹ may be CH₂OH and R² is H. In otherembodiments, one of R¹ and R² is CO₂alkyl and the other is H. Forexample, one of R¹ and R² may be CO₂Et. Thus, R¹ may be H and R² isCO₂Et, or R¹ may be CO₂Et and R² is H. In further embodiments of theinvention, one of R¹ and R² is CO₂H and the other is H. In this case, R¹may be H and R² is CO₂H, or R¹ may be CO₂H and R² is H. In yet furtherembodiments of the invention, one of R¹ and R² may be Me, Et, CH₂OMe, orCH₂OAc.

In some embodiments of the invention, R³ is H. Alternatively, R³ may bealkyl, for example methyl or ethyl, or R³ may be acetyl.

Preferred compounds of the invention include:

The invention also relates to a pharmaceutical composition comprising acompound of the invention and a pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a method of treating cancer,osteoporosis, Type 2 diabetes, or an immune disease comprisingadministering a pharmaceutically effective amount of a compound of theinvention to a patient requiring treatment, or alternatively to the useof a compound of the invention in the manufacture of a medicament fortreating cancer, osteoporosis, Type 2 diabetes, or an immune disease, oralternatively to a pharmaceutical composition for treating cancer,osteoporosis, Type 2 diabetes, or an immune disease, comprising acompound of the invention.

The cancer to be treated by a compound of the invention may be selectedfrom, but is not limited to, leukaemia, ovarian cancer and breastcancer.

The immune disease to be treated by a compound of the invention may beselected from, but is not limited to, asthma, eczema, allergic rhinitis,Type 1 diabetes, rheumatoid arthritis, and lupus.

General Synthesis Methodology

The compounds of the invention may be prepared according to any knowntechniques. The following is a general description of the synthesis ofcompound I. Similar pathways may be employed for preparing othercompounds of the invention.

Referring to Scheme 1, the known glycal 1¹³ may be converted into thebromoalkene 2 via a cyclopropane intermediate that reacts spontaneouslywith sodium acetate. Stereoselective alkyne substitution of the acetatewith bis(trimethylsilyl)acetylene under Lewis acidic conditions with tintetrachloride produces a mixture of the acetonide 3 and diol 4. Afterseparation, acetonide 3 can be converted into 4 by treatment withtrifluoroacetic acid to provide further diol 4. Removal of the silylgroup followed by sodium periodate cleavage affords an aldehyde thatundergoes a Wittig reaction to give the unsaturated ester as a separablemixture of the (Z) isomer 5 and the (E) isomer 6 (4:1 ratio of 5:6).

Each isomer is then individually subjected to the following set ofreactions, as shown in Schemes 2 and 3. Oxymercuration of 5 or 6 affordsthe methyl ketone 7 or 11 and reduction proceeds stereoselectively toprovide the alcohol 8 or 12. Palladium-catalysed carbonylation andlactone formation gives the furo[3,4-b]pyranone 9 or 13. Debenzylationleads to the final compound 10 or 14.

As shown in Scheme 4, the enal 15 can also be derived from diol 4.Oxymercuration affords the ketoaldehyde 16, which is reduced under Lucheconditions to provide the diol 17. Carbonylation and deprotection givesthe diol 19.

As shown in Scheme 5, the alcohol 10 can be converted to the acetate 20.Additionally, the diol 19 can be doubly acetylated to form diacetate 21or monomethylated to give methyl ether 22.

Biological Activity

The compounds of the invention exhibit cytotoxicity and growthinhibition of a range of human, animal and bacterial cell lines, andinhibition of kinases relevant to cancer, diabetes, immune disease orosteoporosis.

Cancer assays were performed using MTT cell proliferation assays withthree different immortalised cell lines: a human leukaemia cell line(HL-60), an ovarian cancer cell line (1A9) and a breast cancer cell line(MCF7).

Kinase inhibition assays were conducted by Life Technologies using theirSelectScreen® Whole Panel ACCESS Biochemical Kinase Profiling Service.The Z'-LYTE® Screening Protocol was used to obtain the results for thefollowing kinases: AMPK A2/B1/G1, BMX, BTK, MAPK14 (p38 alpha), PLK1,TXK. The LanthaScreen Protocol was used to obtain the result for thefollowing kinase: NUAK2.

AMPK A2/B1/G1 refers to an AMP-activated protein kinase (AMPK) which isa heterotrimeric complex that acts as a sensor of cellular energylevels. The signalling cascades initiated by activating AMPK arecritical to regulating metabolic events in the liver, skeletal muscle,heart, adipose tissue, and pancreas. An impairment in fuel metabolismthat occurs in obesity is a factor leading to Type 2 diabetes. Theinsulin resistance associated with Type 2 diabetes is most profound atthe level of skeletal muscle, the primary site of glucose and fatty addutilisation, Activation of AMPK is of interest for the treatment of Type2 diabetes.

Bone marrow tyrosine kinase (BMX) is a non-receptor tyrosine kinase thatbelongs to the Src-related TEC subfamily of tyrosine kinases. It is animportant regulator of various cellular processes including apoptosis,cell survival, cellular differentiation, cell migration, andtransformation, A positive result from a BMX kinase assay is anindicator of potential therapeutic treatment of cancer.

The Src-family kinases are examples of proteins that utiliseautophosphorylation to sustain their activated states. Src kinases areinvolved in intracellular signalling pathways that influence cell growthand cell adhesion strength. The latter contributes to the control ofcell migration. In this way, Src-kinase deregulation can enhance tumourgrowth and invasive potential of cancer cells. Proto-oncogenetyrosine-protein kinase Src also known as proto-oncogene c-Src or simplyc-Src is a non-receptor protein tyrosine kinase protein that in humansis encoded by the SRC gene. This protein phosphorylates specifictyrosine residues in other proteins. An elevated level of activity ofc-Src tyrosine kinase is suggested to be linked to cancer progression bypromoting other signals. Therefore, inhibition of c-Src kinase providesa potential cancer therapy.

MAPK14 refers to a mitogen-activated protein kinase 14 (p38 alpha) whichis a member of the stress-activated protein kinase class of MAPKs,MAPK14 is activated by environmental stresses and cytokines, andrequires phosphorylation (often by MAPK kinase). MAPK14 is involved incell differentiation, proliferation, development and transcriptionregulation. Inhibition of this kinase is relevant to the treatment ofcancer.

NUAK2 is a melanoma oncogene. PLK1 is a proto-oncogene involved in colonand lung cancers. Inhibition of these kinases provide a potential cancertherapy.

Bruton tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase belongingto the SRC-related TEC subfamily of tyrosine kinases. It is involved inprimary immunodeficiency disease. Mutations in the BTK gene have beenlinked to severe developmental blocks in human B-cell ontogeny andimmunodeficiency disorders.

TXK1 is another member of the TEC kinase family. Along with doserelatives BMS, ITK and BTK, this kinase signals downstream of antigenreceptors and other types of receptors. It is involved in T-helper 1(Th1) cytokine production.

Compound 10 was found to be growth inhibitory towards immortalised HL-60(human leukaemia) and MCF7 (breast cancer) cell lines (IC₅₀ 3.6 and 9.0μM, respectively) using MTT assays. This compound also demonstratedinhibition of the following kinases (single-point, percent inhibitionsat 10 μM is given in brackets): BTK (Bruton's tyrosine kinase, 83%),AMPK A2/B1/G1 (81%), PLK1 (81%), BMX (80%), NUAK2 (76%), MAPK14 (p38alpha, 74%), TXK (71%).

Compound 14 was found to be growth inhibitory towards immortalised HL-60(human leukaemia) and 1A9 (ovarian cancer) cell lines (IC₅₀ 2.2 and 3.4μM, respectively) using MTT assays.

Compound 19 was found to be modestly growth inhibitory towardsimmortalised HL-60 (human leukaemia) cell line (IC₅₀ 42 μM) using a MTTassay.

Therefore, compounds 10, 14 and 19 represent potential leads for thetreatment of cancer, Type 2 diabetes and immune disease. The inhibitionof PTH-induced bone resorption by the parent compound TAN-2483B alsoindicates a potential therapeutic application in osteoporosis by thisclass of compounds.

Pharmaceutical Formulations and Administration

The compounds of the invention may be administered to a patient by avariety of routes, including orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, intravenously, intra-muscularly,intra-dermally, subcutaneously or via an implanted reservoir, preferablyintravenously. The amount of compound to be administered will varywidely according to the nature of the patient and the nature and extentof the disorder to be treated. Typically the dosage for an adult humanwill be in the range 50-4800 μg/m² or μg/kg. The specific dosagerequired for any particular patient will depend upon a variety offactors, including the patient's age, body weight, general health, sex,etc.

For oral administration the compounds of the invention can be formulatedinto solid or liquid preparations, for example tablets, capsules,powders, solutions, suspensions and dispersions. Such preparations arewell known in the art as are other oral dosage regimes not listed here.In the tablet form the compounds may be tableted with conventionaltablet bases such as lactose, sucrose and corn starch, together with abinder, a disintegration agent and a lubricant. The binder may be, forexample, corn starch or gelatin, the disintegrating agent may be potatostarch or alginic acid, and the lubricant may be magnesium stearate. Fororal administration in the form of capsules, diluents such as lactoseand dried corn-starch may be employed. Other components such ascolourings, sweeteners or flavourings may be added.

When aqueous suspensions are required for oral use, the activeingredient may be combined with carriers such as water and ethanol, andemulsifying agents, suspending agents and/or surfactants may be used.Colourings, sweeteners or flavourings may also be added.

The compounds may also be administered by injection in a physiologicallyacceptable diluent such as water or saline. The diluent may comprise oneor more other ingredients such as ethanol, propylene glycol, an oil or apharmaceutically acceptable surfactant. In one preferred embodiment, thecompounds are administered by intravenous injection, where the diluentcomprises an aqueous solution of sucrose, L-histidine and apharmaceutically acceptable surfactant, e.g. Tween 20.

The compounds may also be administered topically. Carriers for topicaladministration of the compounds include mineral oil, liquid petrolatum,white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. The compounds may be present asingredients in lotions or creams, for topical administration to skin ormucous membranes. Such creams may contain the active compounds suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include mineral oil, sorbitan monostearate,polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

The compounds may further be administered by means of sustained releasesystems. For example, they may be incorporated into a slowly dissolvingtablet or capsule.

Any reference to prior art documents in this specification is not to beconsidered an admission that such prior art is widely known or formspart of the common general knowledge in the field.

As used in this specification, the words “comprises”, “comprising”, andsimilar words, are not to be interpreted in an exclusive or exhaustivesense. In other words, they are intended to mean “including, but notlimited to”.

The invention is further described with reference to the followingexamples. It will be appreciated that the invention as claimed is notintended to be limited in any way by these examples.

EXAMPLES Example 1 (−)-TAN-2483B Z-ethyl ester (10) Synthesis ofCompound 2

A solution of glycal 1 (493 mg, 1.78 mmol) in a solution of bromoform (5mL) was treated with K₂CO₃ (1.50 g, 10.9 mmol), sodium acetate (540 mg,6.48 mmol) and 18-crown-6 (20 mg, 0.076 mmol). To the resultingsuspension was added tetra-n-butylammonium bromide (93 mg, 0.304 mmol)and the mixture stirred at room temperature for 24 hours. It was thenheated to 82° C. and stirred for two days. The reaction mixture wasfiltered and concentrated to provide a dark-brown liquid. This crudematerial was purified by column chromatography (9:1 petroleumether:ethyl acetate) to obtain the product acetate 2 as an inseparablemixture of anomers and as a colourless oil (364 mg, 47%, 4:1α:β)together with starting material as a yellow oil (69 mg, 14%,contaminated with bromoform). 2: R_(f) 0.18 (10:1 petroleum ether:ethylacetate); ¹H NMR: (CDCl₃) δ=7.38-7.32 (complex m, 5H, Bn), 6.49 (d,J=5.3, 0.2H, H-3), 6.48 (d, J=6.1 Hz, 0.8H, H-3), 6.35 (s, 0.2H, H-1),6.25 (s, 0.8H, H-1), 4.71 (s, 1.6H, PhCH₂), 4.69 (s, 0.4H, PhCH₂), 4.40(m, 1H, H-6), 4.10 (m, 1H, H-7a), 4.04 (dd, J=8.8, 4.7 Hz, 0.8H, H-7b),3.94 (apparent d, J=6.1 Hz, 0.8H, H-4), 3.92 (dd, J=5.8, 2.6 Hz, 0.2H,H-4), 3.89-3.84 (complex m, 0.4H, H-5, H-7b), 3.70 (dd, J=8.5, 2.0 Hz,0.8H, H-5), 2.15 (s, 2.4H, Ac), 2.12 (s, 0.6H, Ac), 1.41 (s, 0.6H, (CH₃)₂C), 1.39 (s, 2.4H, (CH ₃)₂C) 1.38 (s, 0.6H, (CH ₃)₂C) 1.36 (5, 2.4H,(CH ₃)₂C); ¹³C NMR: (CDCl₃) δ 169.5 (C, CH₃CO), 169.2 (C, CH₃CO), 138.0(C, Bn), 137.8 (C, Bn), 131.6 (CH, C-3), 130.2 (CH, C-3), 128.39 (CH,Bn), 128.24 (CH, Bn), 128.06 (CH, Bn), 127.99 (CH, Bn), 127.90 (CH, Bn),127.54 (CH, Bn), 124.6 (CH, C-2), 122.6 (CH, C-2), 109.5 (C, (CH₃)₂C),90.6 (CH, C-1), 90.0 (CH, C-1), 73.3 (broad, 2×CH, 2×C-6), 73.0 (CH,C-5), 72.1 (broad, CH₂ and CH, PhCH₂ and C-5), 71.9 (CH₂, PhCH₂), 69.5(CH, C-4), 69.1 (CH, C-4), 67.1 (CH₂, C-7), 67.0 (CH₂, C-7), 27.01 (CH₃,(CH ₃)₂C), 26.91 (CH₃, (CH₃)₂C), 25.93 (CH₃, (CH₃)₂C), 25.31 (CH₃,(CH₃)₂C), 20.92 (CH₃, Ac), 20.91 (CH₃, Ac); IR (film from CHCl₃) v_(max)1812, 1644, 1604, 1496, 1454, 1328, 1308, 1288, 786, 761, 648 cm⁻¹;HRMS: m/z C₁₉H₂₇ ⁷⁹BrO₆N⁺[M+NH₄]⁺ calcd 444.1016. found 444.1023, m/zC₁₉H₂₇ ⁸¹BrO₆N⁺[M+NH₄]⁺ calcd 446.0998. found 446.1007.

Synthesis of Compounds 3 and 4

Bis(trimethyl)silylacetylene (1.0 mL, 5.11 mmol) and acetate 2 (546 mg,1.27 mmol) were dissolved in dry CH₂Cl₂ (12 mL) and cooled to −78° C.Then SnCl₄ (1.27 mL, 1M solution in CH₂Cl₂) was added dropwise into thecold solution. The combined solution was stirred for 2 hours at the sametemperature and then was quenched with NaHCO₃ (15 mL) and extracted intoCH₂Cl₂ (2×10 mL). The crude mixture containing acetonide 3 and diol 4was purified by gradient column chromatography (20:1 to 2:1 petroleumether:ethyl acetate) to yield 3 (92 mg, 15%) as a colourless oil anddiol 4 (156 mg, 29%) as a colourless oil.

The remaining 3 could be converted into 4 in the following manner.Acetonide-protected alkyne 3 (95 mg, 0.19 mmol) was dissolved in 2 mL ofacetonitrile and cooled to 0° C. using an ice-water bath. Then it wastreated with trifluoroacetic acid (0.5 mL, 6.5 mmol) and slowly warmedup to room temperature. After stirring for 45 min at room temperature,the solution was carefully quenched with powdered NaHCO₃ and dilutedwith distilled water. The organic compounds were extracted into CH₂Cl₂,the organic phase was dried, filtered and concentrated to afford diol 4(81 mg, quantitative) as a colourless oil (combined yield of 4: 44%). 3:R_(f) 0.45 (5:1 petroleum ether:ethyl acetate); [α]_(D) ²²=−45 (c 0.32,CH₂Cl₂)¹H NMR: (CDCl₃) δ=7.37-7.27 (complex m, 5H, Bn), 6.26 (d, J=5.5Hz, 1H, H-5), 4.92 (s, 1H, H-3), 4.69 (s, 2H, PhCH₂), 4.40 (apparent dd,J=13.7, 5.4 Hz 1H, H-9a), 4.15 (dd, J=14.0, 6.6 Hz, 1H, H-9b), 3.97(complex m, 1H, H-8), 3.90 (complex m, J, 2H, H-7, H-6), 1.41 (s, 3H,(CH ₃)₂C), 1.40 (s, 3H, (CH ₃)₂C), 0.18 (s, 9H, (CH ₃)₃Si); ¹³C NMR:(CDCl₃) δ 138.0 (C, Bn), 128.36 (CH, Bn), 128.08 (CH, Bn), 127.84 (CH,Bn), 126.9 (CH, C-5), 124.9 (C, C-4), 109.4 (C, (CH₃)₂ C), 99.4 (C,C-2), 92.3 (C, C-1), 74.2 (CH, C-6), 72.9 (CH, C-8), 72.1 (CH₂, PhCH₂),69.9 (CH, C-3), 69.9 (CH, C-7), 67.6 (CH₂, C-9), 26.8 (CH₃, (CH₃)₂C),25.5 (CH₃, (CH₃)₂C); IR (film from CH₂Cl₂) v_(max) 2986, 2959, 2873,1643, 1455, 1297, 1106, 1079, 1212, 906, 841, 731, 697 cm⁻¹; HRMS: m/zC₂₂H₃₀ ⁸¹BrO₄Si⁺[M+H]⁺ calcd 467.1074. found 467.1056. 4: R_(f) 0.38(2:1 petroleum ether:ethyl acetate); [α]_(D) ²¹=−100 (c 0.2, CH₂Cl₂); ¹HNMR: (CDCl₃) δ=7.38-7.31 (complex m, 5H, Bn), 6.37 (d, J=5.4 Hz, 1H,H-5), 4.95 (s, 1H, H-3), 4.73 (d, J=11.7 Hz, 1H, PhCH₂), 4.59 (d, J=11.7Hz, 1H, PhCH₂), 4.02-3.96 (complex m, 3H, H-6, H-7, H-8), 4.01-3.84(apparent d, J=11.6 Hz, H, H-9a), 3.78 (dd, J=11.6, 4.6 Hz, 1H, H-9b),0.18 (s, 9H, (CH₃)₃Si); ¹³C NMR: (CDCl₃) δ 137.6 (C, Bn), 128.69 (CH,Bn), 128.55 (CH, Bn), 128.19 (CH, Bn), 126.4 (CH, C-5), 125.5 (CH, C-4),99.3 (C, C-2), 92.4 (C, C-1), 72.5 (CH, C-6), 71.2 (CH₂, PhCH₂), 69.93(CH, C-7 or C-8 or C-3), 69.76 (CH, C-7 or C-8 or C-3), 69.38 (CH, C-7or C-8 or C-3) 64.0 (CH₂, C-9); IR (film from CH₂Cl₂) v_(max) 3486,2985, 2873, 1644, 1454, 1297, 1106, 1079, 912, 843, 732, 698 cm⁻¹; HRMS:m/z C₁₉H₂₅ ⁸¹BrO₄SiNa⁺[M+Na]⁺ calcd 449.0580. found 449.0575.

Synthesis of Compounds 5 and 6

Diol 4 (270 mg, 0.63 mmol) was dissolved in dichloromethane (7.5 mL) andtreated with MeOH (1.5 mL). The reaction mixture became a whitesuspension and was stirred until the reaction was deemed complete (2hours). This mixture was diluted with CH₂Cl₂ and filtered into a brinesolution. The aqueous layer was extracted with CH₂Cl₂ (2×10 mL) and thecombined organic fractions evaporated at room temperature under reducedpressure to afford the diol-containing terminal alkyne as a white solid.The product was used in the next reaction without further purification.R_(f) 0.29 (1:1 petroleum ether:ethyl acetate); m.p. 147.9-148.8° C.;[α]_(D) ²²=−100 (c 0.1, CH₂Cl₂); ¹H NMR: (CDCl₃) δ=7.39-7.31 (complex m,5H, Bn), 6.42 (d, J=5.4 Hz, 1H, H-5), 5.00 (s, 1H, H-3), 4.74 (d, J=11.8Hz, 1H, PhCH₂), 4.60 (d, J=11.7 Hz, 1H, PhCH₂), 4.03 (dd, J=5.6, 2.2 Hz,1H, H-6), 3.99-3.79 (complex m, 2H, H-7, H-8), 3.86-3.84 (apparent d,J=10.6 Hz, 1H, H-9a), 3.78 (dd, J=10.9, 5.2 Hz, 1H, H-9b), 2.53 (d,J=2.2 Hz, 1H, H-1); ¹³C NMR: (CDCl₃) δ 137.6 (C, Bn), 128.69 (CH, Bn),128.22 (CH, Bn), 128.04 (CH, Bn), 126.7 (CH, C-5), 125.1 (CH, C-4), 78.2(C, C-2), 75.0 (C, C-1), 72.5 (CH, C-7 or C-8), 71.3 (CH₂, PhCH₂), 69.8(CH, C-7 or C-8), 69.4 (CH, C-6), 69.2 (CH, C-3) 63.8 (CH₂, C-9); IR(film from CH₂Cl₂) v_(max) 3486, 3088, 2981, 1496, 1455, 1369, 1339,1252, 1214, 1129, 987, 848 cm⁻¹; HRMS: m/z C₁₆H₁₈ ⁸¹BrO₄ ⁺[M+H]⁺ calcd353.0388. found 353.0367.

A solution of the crude diol described above (120 mg, 0.34 mmol) in THF(10 mL) and pH 7 phosphate buffer (3 mL) was treated with NaIO₄ (508 mg,2.38 mmol) in one portion. The resulting mixture was stirred for 1 hour,diluted with brine (50 mL), and extracted with Et₂O (3×50 mL). Thecombined organic layers were washed with brine, dried over MgSO₄ andevaporated to give a colourless oil, which was used without furtherpurification. Next the crude aldehyde product was dissolved in THF (10mL) and treated with ethyl triphenylphosphoranylidene acetate (236 mg,0.68 mmol). The solution was stirred overnight before concentratingunder reduced pressure and purifying with column chromatography (14:1petroleum ether:ethyl acetate) to yield both (Z)-isomer 5 (94 mg, 38%)and (E)-isomer 6 (25 mg, 10%) as colourless oils (combined yield: 48%over three steps). 5: R_(f) 0.33 (14:1 petroleum ether:ethyl acetate);[α]_(D) ²⁰=−188 (c 0.26, CH₂Cl₂); ¹H NMR: (CDCl₃) δ=7.33-7.26 (complexm, 5H, Bn), 6.35 (dd, J=5.4, 1.2 Hz, 1H, H-5), 6.33 (dd, J=11.3, 6.8 Hz,1H, H-8), 5.93 (d, J=11.3 Hz, 1H, H-9), 5.55 (broad d, J=6.6 Hz, 1H,H-7), 5.02 (s, 1H, H-3), 4.58 (d, J=11.4 Hz, 1H, PhCH₂), 4.50 (d, J=11.7Hz, 1H, PhCH₂), 4.20 (dd, J=5.6, 2.7 Hz 1H, H-6), 4.14 (q, J=7.2 Hz, 2H,OEt), 2.50 (s, 1H, H-1), 1.28 (t, J=7.2 Hz, 3H, OEt); ¹³C NMR: (CDCl₃) δ165.6 (C, C-10), 144.8 (CH, C-8), 137.5 (C, Bn), 128.40 (CH, Bn), 128.2(CH, C-5), 127.98 (CH, Bn), 127.89 (CH, Bn), 120.5 (C, C-9), 78.1 (C,C-2), 75.1 (CH, C-1), 71.8 (CH₂, Bn), 71.3 (CH, C-6), 71.0 (CH, C-7),68.8 (CH, C-3), 60.4 (CH₂, OEt), 14.2 (CH₃, OEt); IR (film from CH₂Cl₂)v_(max) 3031, 2931, 2872, 2116, 1648, 1539, 1454, 1497, 1388, 1366,1334, 1031, 911, 846, 735, 698 cm⁻¹; HRMS: m/z C₁₉H₂₁ ⁸¹BrO₄ ⁺[M+H]⁺calcd 393.0521. found 393.0533. 6: R_(f) 0.45 (2:1 petroleum ether:ethyl acetate); [α]_(D) ²⁰=−112 (c 0.34, CH₂Cl₂); ¹H NMR: (CDCl₃) δ7.37-7.26 (complex m, 5H, Bn), 7.02 (dd, J=15.8, 4.2 Hz, 1H, H-8), 6.34(d, J=5.5 Hz, 1H, H-5), 6.22 (dd, J=15.8, 1.5 Hz, 1H, H-9), 5.09 (s, 1H,H-3), 4.73 (m, 1H, H-7), 4.61 (d, J=11.8 Hz, 1H, PhCH₂), 4.53 (d, J=11.8Hz, 1H, PhCH₂), 4.23 (q, J=7.1 Hz, 2H, OEt), 3.92 (dd, J=5.3, 2.8 Hz,1H, H-6), 2.53 (d, J=2.1 Hz, 1H, H-1), 1.31 (t, J=7.1 Hz, 3H, OEt); ¹³CNMR: (CDCl₃) δ 166.0 (C, C-10), 142.5 (CH, C-8), 137.4 (C, Bn), 128.13(CH, Bn), 128.03 (CH, Bn), 127.99 (CH, Bn), 126.6 (CH, C-5), 124.9 (C,C-4), 122.7 (CH, C-9), 77.9 (C, C-2), 75.3 (CH, C-1), 72.2 (CH, C-7),71.1 (CH₂, PhCH₂), 71.0 (CH, C-6), 60.5 (CH₂, OEt), 14.6 (CH₃, OEt); IR(film from CH₂Cl₂) v_(max) 2360, 2115, 1664, 1496, 1454, 1392, 1368,898, 847, 798, 645, 631, 603 cm⁻¹; HRMS: m/z C₁₉H₂₃ ⁸¹BrO₄N⁺[M+NH₄]⁺calcd 408.0805. found 408.0819.

Synthesis of Compound 7

A sample of Z-ethyl ester 5 (125 mg, 0.32 mmol) was dissolved in THF(1.5 mL). HgSO₄ in H₂SO₄ (10% aqueous solution, 1.5 mL, 19 mg, 0.2equiv) was added to this solution. The reaction mixture was stirred atroom temperature for 16 hours. The mixture was diluted with Et₂O (5 mL)and carefully neutralised with NaHCO₃ powder until pH=7 was reached. Theaqueous layer was washed with Et₂O (3×5 mL). The organic layers werecombined and dried over MgSO₄. The crude product was chromatographed(9:1 petroleum ether:ethyl acetate) to yield the product 7 as a clearoil (95 mg, 72%). 7: R_(f) 0.31 (9:1 petroleum ether:ethyl acetate);[α]_(D) ²¹=−133 (c 0.14, CH₂Cl₂); ¹H NMR: (CDCl₃) δ 7.34-7.28 (complexm, 5H, Bn), 6.48 (d, J=5.2 Hz, 1H, H-5), 6.36 (dd, J=11.6, 7.2 Hz, 1H,H-8), 5.94 (d, J=11.9 Hz, 1H, H-9), 5.31 (broad d, J=6.9 Hz, 1H, H-7),4.74 (s, 1H, H-3), 4.59 (d, J=11.9 Hz, 1H, PhCH₂), 4.52 (d, J=11.7 Hz,1H, PhCH₂), 4.17 (dd, J=5.0, 3.0 Hz, 1H, H-6), 4.13 (q, J=7.1 Hz, 2H,OEt), 2.32 (s, 3H, H-1), 1.27 (t, J=7.1 Hz, 3H, OEt); ¹³C NMR: (CDCl₃) δ202.8 (C, C-2), 165.3 (C, C-10), 144.5 (CH, C-8), 137.5 (C, Bn), 128.43(CH, C-5), 128.40 (CH, Bn), 128.04 (CH, Bn), 127.97 (CH, Bn), 121.50 (C,C-9), 81.2 (CH, C-3), 71.8 (CH₂, Bn), 71.4 (CH, C-6), 71.2 (CH, C-7),60.5 (CH₂, OEt), 28.1 (CH₃, C-1), 14.1 (CH₃, OEt); IR (film from CH₂Cl₂)v_(max) 3063, 2872, 1649, 1496, 1454, 1388, 1302, 979, 697 cm⁻¹; HRMS:m/z C₁₉H₂₅BrO₅N⁺[M+NH₄]⁺ calcd 426.0911. found 426.0902.

Synthesis of Compound 8

A sample of methyl ketone 7 (95 mg, 0.23 mmol) was dissolved in MeOH (2mL) and cooled to −78° C. using an acetone-dry ice bath and treated withNaBH₄ (9 mg, 0.28 mmol). The solution was stirred for 45 minutes at thesame temperature to complete the reaction. Unreacted NaBH₄ was quenchedwith acetone (1 mL). The solvent was removed under reduced pressure andthe residue redissolved in dichloromethane. The organic phase was washedwith water. The extracted organic layer was evaporated and purified bycolumn chromatography (5:1 petroleum ether:ethyl acetate) to yieldproduct 8 as a colourless oil (85 mg, 91%). 8: R_(f) 0.24 (5:1 petroleumether:ethyl acetate); [α]_(D) ²⁰=−65 (c 0.2, CH₂Cl₂). ¹H NMR: (CDCl₃)δ=7.34-7.28 (complex m, 5H, Bn), 6.48 (dd, J=4.8, 1.4 Hz, 1H, H-5), 6.44(dd, J=11.7, 7.8 Hz, 1H, H-8), 5.99 (d, J=11.8 Hz, 1H, H-9), 5.80 (ddd,J=7.6, 3.2, 1.6 Hz, 1H, H-7), 4.62 (d, J=12.0 Hz, 1H, PhCH₂), 4.55 (d,J=12.0 Hz, 1H, PhCH₂), 4.36 (m, 1H, H-2), 4.16 (q, J=7.1 Hz, 2H, OEt),4.12 (partially obscured dd, J=4.2, 3.4 Hz, 1H, H-6), 4.05 (apparent s,1H, H-3), 1.34 (d, 3H, J=6.9 Hz, H-1), 1.28 (t, J=7.2 Hz, 3H, OEt); ¹³CNMR: (CDCl₃) δ 165.7 (C, C-10), 144.3 (CH, C-8), 137.8 (C, Bn), 129.2(CH, C-5), 128.39 (CH, Bn), 128.87 (CH, Bn), 127.84 (CH, Bn), 125.2 (C,C-4), 122.5 (CH, C-9), 79.5 (CH, C-3), 71.8 (CH, C-6), 71.4 (CH₂,PhCH₂), 69.9 (CH, C-7), 68.0 (CH, C-2), 60.5 (CH₂, OEt), 19.6 (CH₃,C-1), 14.1 (CH₃, OEt); IR (film from CH₂Cl₂) v_(max) 3467, 3031, 2980,2930, 1650, 1454, 1232, 1147, 856, 827, 697 cm⁻¹; HRMS: m/z C₁₉H₂₅O₅⁺[M+H]⁺ calcd 333.1697. found 333.1686.

Synthesis of Compound 9

Alcohol 8 (42 mg, 0.11 mmol) was dissolved in 1,4-dioxane (2 mL). ThenXantPhos (15 mg, 0.025 mmol, 20 mol %), palladium acetate (6 mg, 0.025mmol, 20 mol %), sodium carbonate (67 mg, 0.63 mmol), TBAI (9 mg, 0.025mmol) and triethylamine (0.18 ml, 1.26 mmol) were added sequentially. COgas was bubbled through the reaction mixture for a few minutes tosaturate the solution with CO. Then the flask was connected to a refluxcondenser under an atmosphere of CO (balloon). The whole set-up wasevacuated and purged with CO three times and stirred vigorously at 95°C. overnight. Another portion of XantPhos and palladium acetate (10 mol% each) was added after 16 hours and the mixture stirred vigorously at95° C. under CO until the reaction was complete (a further 3 hours). Themixture was diluted with dichloromethane and filtered through a silicaplug. The crude product was purified by column chromatography (3:1petroleum ether:ethyl acetate) to yield the lactone 9 as a colourlessoil (22 mg, 60%). 9: R_(f) 0.37 (5:1 petroleum ether: ethyl acetate);[α]_(D) ²⁰=−178 (c 0.18, CH₂Cl₂); ¹H NMR: (CDCl₃) δ=7.35-7.28 (complexm, 5H, Bn), 7.22 (dd, J=5.4, 3.2 Hz, 1H, H-5), 6.49 (dd, J=11.5, 7.7 Hz,1H, H-8), 5.99 (dd, J=11.5, 1.0 Hz, 1H, H-9), 5.28 (m, 1H, H-7), 5.14(dd, J=7.5, 3.2 Hz, 1H, H-3), 4.85 (apparent q, J=6.8 Hz, 2H, H-2), 4.60(d, J=11.9 Hz, 1H, PhCH₂), 4.51 (d, J=12.2 Hz, 1H, PhCH₂), 4.42 (dd,J=5.4, 2.9 Hz 1H, H-6), 4.15 (q, J=7.2 Hz, 2H, OEt), 1.29-1.26 (apparentm, 6H, OEt, C-1); ¹³C NMR: (CDCl₃) δ 166.4 (C, C-13), 165.6 (C, C-10),145.5 (CH, C-8), 137.4.0 (C, Bn), 134.9 (CH, C-5), 132.3 (C, C-4),128.48 (CH, Bn), 128.07 (CH, Bn), 127.80 (CH, Bn), 122.3 (CH, C-9), 78.4(CH, C-2), 71.8 (CH₂, PhCH₂), 71.6 (CH, C-7), 71.2 (CH, C-3), 69.5 (CH,C-6), 60.5 (CH₂, OEt), 15.4 (CH₃, OEt or 1), 14.2 (CH₃, OEt or C-1); IR(film from CH₂Cl₂) v_(max) 2983, 1650, 1454, 1413, 1386, 1298, 1096,920, 829, 736, 689, 632 cm⁻¹; HRMS: m/z C₂₃H₂₃O₆ ⁺[M+H]⁺ calcd 362.1595.found 362.1575.

Synthesis of Compound 10

To a solution of lactone 9 (30.0 mg, 0.08 mmol) in CH₂Cl₂ (1 mL) wasadded TiCl₄ (27 μL, 0.25 mmol) in CH₂Cl₂ (0.1 mL) at 0° C. Afterstirring at the same temperature for 10 min, the reaction was quenchedwith saturated aqueous NaHCO₃ (5 mL), and the organic layer wasseparated and extracted with CH₂Cl₂ (2×5 mL). The organic layers werecombined and dried over anhydrous MgSO₄. After filtration andconcentration under reduced pressure, the crude product was purified bycolumn chromatography (2:1 petroleum ether: ethyl acetate) to yield(Z)-TAN-2483B ethyl ester 10 as a colourless oil (13 mg, 56%). 10: R_(f)0.17 (2:1 petroleum ether:ethyl acetate); ¹H NMR: (CDCl₃) δ=7.16 (dd,J=5.4, 3.4 Hz, 1H, H-5), 6.44 (dd, J=11.7, 7.8 Hz, 1H, H-8), 6.07 (dd,J=11.7, 1.2 Hz, 1H, H-9, 5.32 (ddd, J=7.8, 3.4, 1.4 Hz, 1H, H-7), 5.12(ddd, J=5.4, 3.2, 1.3 Hz, 1H, H-3), 4.85 (quintet, J=6.8 Hz, 2H, H-2),4.71 (dd, J=4.8, 3.6 Hz 1H, H-6), 4.19 (q, J=7.2 Hz, 2H, OEt), 1.32-1.28(apparent m, 6H, OEt, H-1); ¹³C NMR: (CDCl₃) δ 166.4 (C, C-10), 166.1(C, C-13), 143.7 (CH, C-8), 135.4 (CH, C-5), 130.9 (C, C-4), 123.3 (CH,C-9), 78.0 (CH, C-2), 72.9 (CH, C-3), 70.4 (CH, C-7), 69.6 (CH, C-6),60.9 (CH₂, OEt), 15.2 (CH₃, OEt or C-1), 14.1 (CH₃, OEt or C-1), IR(film from CH₂Cl₂) v_(max) 3572, 3458, 1650, 1447, 1414, 1386, 1331,1301, 1121, 1042, 968, 917, 852, 822, 764, 731 cm⁻¹; HRMS: m/z C₁₃H₁₇O₆⁺[M+H]⁺ calcd 268.0947. found 269.1012.

Example 2 (−)-TAN-2483B E-ethyl ester (14) Synthesis of Compound 11

A sample of alkyne 6 (35 mg, 0.09 mmol) was dissolved in THF (0.7 mL).HgSO₄ (5 mg, 0.2 equiv, in 0.7 mL of 10% H₂SO₄ solution) was added tothis solution. Then this solution was stirred at room temperature untilthe starting material disappeared (overnight). The mixture was dilutedwith Et₂O (5 mL) and carefully neutralised with powdered NaHCO₃ untilpH=7. The aqueous layer was washed with Et₂O (3×5 mL). The organiclayers were combined and dried over MgSO₄. After filtering andconcentrating under reduced pressure, the crude product waschromatographed (5:1 petroleum ether:ethyl acetate) to yield the product11 as clear oil (22 mg, 61%). 11: R_(f) 0.25 (5:1 petroleum ether:ethylacetate); [α]_(D) ²³=−224 (c 0.45, CH₂Cl₂); ¹H-NMR: (CDCl₃) δ_(H)7.36-7.29 (complex m, 5H, Bn), 6.98 (dd, J=15.8, 4.2 Hz, 1H, H-9), 6.50(dd, J=4.1, 0.7 Hz, 1H, H-5), 6.19 (dd, J=15.8, 1.4 Hz, 1H, H-8), 4.81(s, 1H, H-3), 4.62 (d, J=11.7 Hz, 1H, PhCH₂), 4.54 (d, J=12.0 Hz, 1H,PhCH₂), 4.47 (m, 1H, H-7), 4.24 (q, J=7.2 Hz, 2H, OEt), 3.95 (dd, J=4.3,3.8 Hz, 1H, H-6), 2.33 (s, 3H, H-1), 1.32 (t, J=7.1 Hz, 3H, OEt);¹³C-NMR: (CDCl₃) δ_(C) 202.6 (C, C-2), 165.3 (C, C-10), 142.1 (CH, C-9),137.3 (C, Bn), 128.53 (CH, Bn), 128.52 (CH, Bn), 128.09 (CH, Bn), 127.99(CH, C-5), 123.1 (CH, C-8), 121.2 (C, C-4), 80.8 (CH, C-3), 72.8 (CH,C-7), 71.2 (CH, C-6 and CH₂, PhCH₂), 60.6 (CH₂, OEt), 28.1 (CH₃, C-1),14.2 (CH₃, OEt); IR (film from Et₂O) v_(max) 3063, 1664, 1642, 1604,1496, 1419, 924, 866, 612 cm⁻¹; HRMS: m/z C₁₉H₂₅ ⁸¹BrO₅N⁺[M+NH₄]⁺ calcd426.0911. found 426.0929.

Synthesis of Compound 12

A sample of methyl ketone 11 (22 mg, 0.05 mmol) was dissolved in MeOH (1mL), cooled to −78° C. and treated with NaBH₄ (2 mg, 0.06 mmol). Thesolution was stirred for 15 minutes at same temperature to complete thereaction. Unreacted NaBH₄ was quenched with acetone (1 mL). Solventswere removed under reduced pressure, redissolved in CH₂Cl₂ (10 mL) andwashed with distilled water (10 mL). The aqueous layer was extractedwith CH₂Cl₂ (2×10 mL). Organic fractions were combined, evaporated andpurified by column chromatography (5:1 petroleum ether:ethyl acetate) toyield compound 12 as a colourless oil (19 mg, 91%). 12: R_(f) 0.14 (5:1petroleum ether:ethyl acetate); [α]_(D) ²¹=−137 (c 0.15, CH₂Cl₂);¹H-NMR: (CDCl₃) δ_(H) 7.37-7.27 (complex m, 5H, Bn), 7.03 (dd, J=15.8,4.3 Hz, 1H, H-8), 6.46 (dd, J=3.8, 1.6 Hz, 1H, H-5), 6.20 (d, J=15.7 Hz,1H, H-9), 4.95 (m, 1H, H-7), 4.61 (d, J=12.1 Hz, 2H, PhCH₂), 4.56 (d,J=12.0 Hz, 2H, PhCH₂), 4.39 (m, 1H, H-3), 4.23 (q, J=7.0 Hz, 2H, OEt),4.14-4.06 (complex m, 2H, H-2 and H-6), 1.32-1.24 (complex m, 6H, H-1and OEt); ¹³C-NMR: (CDCl₃) δ_(C) 166.2 (C, C-10), 142.8 (CH, C-8), 137.5(C, Bn), 129.5 (CH, C-5), 128.49 (CH, Bn), 128.08 (CH, Bn), 127.88 (CH,Bn), 124.1 (C, C-4), 123.2 (CH, C-9), 78.5 (CH, C-2), 72.9 (CH, C-7),72.0 (CH, C-6), 71.1 (CH₂, PhCH₂), 68.0 (CH, C-3), 60.7 (CH₂, OEt), 19.3(CH₃, C-1), 14.1 (CH₃, OEt); IR (film from CH₂Cl₂) v_(max) 3572, 3458,2983, 2926, 1650, 1447, 1414, 1331, 1121, 1042, 968, 852, 822, 764, 731cm⁻¹; HRMS: m/z C₁₉H₂₇ ⁸¹BrNO₅ ⁺[M+NH₄]⁺ calcd 428.1073. found 428.1078.

Synthesis of Compound 13

A sample of E-ethyl ester 12 (20 mg, 0.05 mmol) was dissolved in1,4-dioxane (1 mL). Then XantPhos (5.6 mg, 9.72×10⁻³ mmol, 20%),palladium acetate (2 mg, 9.72×10⁻³ mmol, 20%), sodium carbonate (25 mg,0.24 mmol), TBAI (3 mg, 9.72×10⁻³ mmol) and triethylamine (68 μL, 0.49mmol) were added accordingly. This mixture was bubbled with CO for a fewminutes to saturate the solution with CO. Then the flask was connectedto a reflux condenser and placed under an atmosphere of CO (balloon).The whole set-up was evacuated and purged with CO three times andstirred vigorously at 95° C. for 2.5 hours. Then the solution wasdiluted with dichloromethane (5 mL) and filtered through a silica plug.The crude product was purified by column chromatography (3:1 petroleumether: ethyl acetate) to yield lactone 13 as a colourless oil (8 mg,46%). 13: R_(f) 0.24 (3:1 petroleum ether:ethyl acetate); [α]_(D)²⁰=−234 (c 0.3, CHCl₃); ¹H-NMR: (CDCl₃) δ_(H) 7.38-7.29 (complex m, 5H,Bn), 7.13 (dd, J=4.3, 3.7 Hz, 1H, H-5), 7.06 (dd, J=15.8, 5.4 Hz, 1H,H-8), 6.23 (dd, J=15.9, 1.2 Hz, 1H, H-9), 5.08 (m, 1H, H-3), 4.86(quintet, J=6.8 Hz, 1H, H-2), 4.65 (d, J=12.2 Hz, 2H, PhCH₂), 4.54 (d,J=12.2 Hz, 2H, PhCH₂), 4.39 (m, 1H, H-7), 4.28 (apparent t, J=3.6 Hz 1H,H-6), 4.24 (q, J=7.1 Hz, 2H, OEt), 1.32 (t, J=7.2 Hz, 3H, OEt), 1.24 (d,J=6.6 Hz, 3H, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 166.0 (C, C-10), 165.8 (C,C-13), 141.9 (CH, C-8), 137.0 (C, Bn), 134.2 (CH, C-5), 132.2 (C, C-4),128.63 (CH, Bn), 128.27 (CH, Bn), 127.85 (CH, Bn), 124.9 (CH, C-9), 77.9(CH, C-2), 74.3 (CH, C-7), 71.8 (CH₂, PhCH₂), 70.5 (CH, C-3), 69.5 (CH,C-6), 60.7 (CH₂, OEt), 15.3 (CH₃, C-1), 14.2 (CH₃, OEt); IR (film fromEt₂O) v_(max) 2980, 2959, 2926, 1714, 1662, 1447, 1439, 1384, 1302,1266, 1180, 1039, 977, 915, 825, 726, 694, 666 cm⁻¹; HRMS: m/z C₂₀H₂₆NO₆⁺[M+NH₄]⁺ calcd 376.1760. found 376.1768.

Synthesis of Compound 14

To a solution of benzyl-protected furopyrone 13 (8.0 mg, 0.02 mmol) inCH₂Cl₂ (1 mL) was added TiCl₄ (7.4 μL, 0.07 mmol) in CH₂Cl₂ (0.1 mL) at0° C. After stirring at the same temperature for 10 min, the reactionwas quenched with saturated aqueous NaHCO₃ (5 mL), and the organic layerwas separated and extracted with CH₂Cl₂ (2×5 mL). The organic layerswere combined and dried over anhydrous MgSO₄. After filtration andconcentration under reduced pressure, the crude product was purified bycolumn chromatography (2:1 petroleum ether:ethyl acetate) to yield, as apale yellow oil, (E)-TAN-2483B ethyl ester 14 (3 mg, 56%). 14: R_(f)0.33 (1:1 petroleum ether:ethyl acetate); [α]_(D) ²²=−100 (c 0.1, Et₂O);¹H-NMR: (CDCl₃) δ_(H) 7.17 (dd, J=5.6, 3.3 Hz, 1H, H-5), 7.02 (dd,J=15.7, 5.2 Hz, 1H, H-8), 6.29 (dd, J=15.7, 1.7 Hz, 1H, H-9), 5.13 (dd,J=7.7, 3.1 Hz, 1H, H-3), 4.81 (quintet, J=7.0 Hz, 1H, H-2), 4.56 (m, 1H,H-6), 4.41 (m, 1H, H-7), 4.23 (q, J=7.1 Hz, 2H, OEt), 1.31 (t, J=7.2 Hz,3H, OEt), 1.28 (d, J=6.6 Hz, 3H, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 165.9 (C,C-13), 165.8 (C, C-10), 141.2 (CH, C-8), 134.8 (CH, C-5), 131.6 (C,C-4), 125.4 (CH, C-9), 77.9 (CH, C-2), 75.1 (CH, C-7), 70.8 (CH, C-3),63.5 (CH, C-6), 60.8 (CH₂, OEt), 15.3 (CH₃, C-1), 14.2 (CH₃, OEt); IR(film from CDCl₃) v_(max) 3760, 3672, 3644, 3447, 2983, 2929, 1663,1448, 1370, 1096, 980, 942, 916, 869, 826, 730, 699, 667 cm⁻¹; HRMS: m/zC₁₃H₂₀O₆N⁺[M+NH₄]⁺ calcd 286.1285. found 286.1290.

Example 3 Hydroxy-TAN-2483B (19) Synthesis of Compound 15

Diol 4 (58 mg, 0.14 mmol) was dissolved in a solution of 20% v/vmethanol in dichloromethane (5 mL) and treated with potassium carbonate(94 mg, 0.68 mmol). The solution was stirred at room temperature untilthe reaction was complete according to TLC analysis. The reactionmixture was filtered, treated with brine (10 mL) and extracted withdichloromethane (3×5 mL). The organic fractions were combined, driedover anhydrous MgSO₄ and evaporated quickly on rotary evaporator (Note:the water bath temperature of the rotary evaporator must be kept closeto room temperature to avoid degradation). The crude diol wasredissolved in 5 mL of THF and treated with NaIO₄ (145 mg, 0.68 mmol)and stirred one hour at room temperature. Reaction was quenched with 5mL of brine after starting material has been completely consumed. Thenit was extracted into diethyl ether (3×5 mL) and organic layer was driedand evaporated on rotary evaporator. The crude aldehyde was redissolvedin dry THF (2 mL) and treated with(triphenylphosphoranylidene)acetaldehyde (42 mg, 0.13 mmol) and stirredovernight at room temperature. The excess solvents in the crude mixturewere evaporated and the residue purified by column chromatography (5:1petroleum ether:ethyl acetate) to obtain 15 as a colourless oil (20 mg,42% over three steps). 15: R_(f) 0.18 (5:1 petroleum ether: ethylacetate); [α]_(D) ²¹=−98 (c 1.7, CH₂Cl₂); ¹H NMR: (CDCl₃) δ_(H) 9.60 (d,J=7.5 Hz, 1H, H-10), 7.38-7.27 (complex m, 5H, Bn), 6.81 (dd, J=15.9,4.1 Hz, 1H, H-8), 6.44 (ddd, J=15.8, 7.8, 1.9 Hz, 1H, H-9), 6.39 (dd,J=5.4, 1.2 Hz, 1H, H-5), 5.12 (d, J=1.7 Hz, 1H, H-3), 4.83 (complex m,1H, H-7), 4.63 (d, J=11.9 Hz, 1H, PhCH₂), 4.50 (d, J=11.7 Hz, 1H,PhCH₂), 3.99 (dd, J=5.4, 3.0 Hz, 1H, H-6), 2.55 (d, J=2.1 Hz, 1H, H-1);¹³C NMR: (CDCl₃) δ_(C) 192.9 (CH, C-10), 151.0 (CH, C-8), 137.2 (C, Bn),132.7 (CH, C-9), 128.59 (CH, Bn), 128.21 (CH, Bn), 128.00 (CH, Bn),126.3 (CH, C-5), 125.1 (C, C-4), 77.7 (CH, C-2), 75.6 (C, C-1), 72.2(CH, C-7), 70.8 (CH₂, PhCH₂ and CH, C-6), 69.1 (CH, C-3); IR (film fromCDCl₃) v_(max) 2733, 2217, 2154, 1722, 1646, 1496, 1367, 1301, 1209, 848cm⁻¹; HRMS: m/z C₁₇H₂₁ ⁸¹BrO₃N⁺[M+NH₄]⁺ calcd 364.0543. found 364.0528.

Synthesis of Compound 16

A sample of alkyne 15 (20 mg, 0.06 mmol) was dissolved in THF (0.5 mL).HgSO₄ in H₂SO₄ (10% aqueous solution, 0.5 mL, 3 mg, 0.012 mmol) wasadded to this solution. The reaction mixture was stirred at roomtemperature for 4 hours. The mixture was diluted with Et₂O (5 mL) andcarefully neutralized with NaHCO₃ powder until pH=7 was reached. Theaqueous layer was washed with Et₂O (3×5 mL). The organic layers werecombined and dried over MgSO₄. The crude product was chromatographed(3:1 petroleum ether:ethyl acetate) to yield the product 16 as a clearoil (16 mg, 76%). 16: R_(f) 0.37 (3:1 petroleum ether:ethyl acetate);[α]_(D) ¹⁹=−126.9 (c 0.52, CH₂Cl₂); ¹H NMR: (CDCl₃) δ_(H) 9.58 (d, J=7.6Hz, 1H, H-10), 7.37-7.27 (complex m, 5H, Bn), 6.77 (dd, J=15.8, 4.1 Hz,1H, H-8), 6.53 (dd, J=4.9, 1.4 Hz, 1H, H-5), 6.42 (ddd, J=16.0, 7.8, 2.0Hz, 1H, H-9), 4.84 (s, H, 1H, H-3), 4.63 (complex m, J=11.9 Hz, 2H,PhCH₂ and H-7), 4.52 (d, J=12.0 Hz, 1H, PhCH₂), 4.06 (dd, J=4.9, 1.7 Hz,1H, H-6), 2.34 (s, 3H, H-1); ¹³C NMR: (CDCl₃) δ_(C) 202.4 (C, C-2),192.9 (CH, C-10), 150.6 (CH, C-8), 137.1 (C, Bn), 132.9 (CH, C-9),128.62 (CH, Bn), 128.29 (CH, Bn), 128.0 (CH, Bn), 127.0 (CH, C-5), 121.3(C, C-4), 80.7 (CH, C-3), 72.8 (CH, C-7), 71.1 (CH, C-6 and CH₂, PhCH₂),28.4 (CH₃, C-1); IR (film from CDCl₃) v_(max) 2958, 2930, 2871, 2733,2360, 2341, 1881, 1648, 1551, 1496, 1418, 1273, 1208, 1170, 1144, 861,790, 620 cm⁻¹; HRMS: m/z C₁₇H₂₁ ⁸¹BrO₄N⁺[M+NH₄]⁺ calcd 382.0654. found382.0646.

Synthesis of Compound 17

CeCl₃.7H₂O (16 mg, 0.0301 mmol) was added to a solution of ketoaldehyde16 (16 mg, 0.0301 mmol) in CH₂Cl₂/EtOH (0.6 mL of a 1:1 v/v mixture)maintained at room temperature. This mixture was then cooled to −78° C.,treated with NaBH₄ (5.8 mg, 0.15 mmol) in EtOH (0.3 mL) and stirringcontinued at −78° C. for one hour. After this time TLC analysis (1:3ethyl acetate: petroleum ether) showed the absence of starting material.Then 0.5 mL of acetone was added, concentrated under reduced pressure togive yellow oil. This material was purified by flash chromatography (1:1petroleum ether:ethyl acetate) to obtain 17 as a colourless oil (14 mg,93%). 17: R_(f) 0.24 (1:1 petroleum ether:ethyl acetate); [α]_(D)²⁴=−16.6 (c 0.15, CH₂Cl₂); ¹H-NMR: (CDCl₃) δ_(H) 7.36-7.28 (complex m,5H, Bn), 6.42 (dd, J=3.4, 1.8 Hz, 1H, H-5), 6.07 (dt, J=15.7, 5.3 Hz,1H, H-9), 5.93 (dd, J=15.9, 6.4 Hz, 1H, H-8), 4.75 (apparent t, J=5.4Hz, 1H, H-7), 4.62 (d, J=11.9 Hz, 2H, PhCH₂), 4.56 (d, J=11.9 Hz, 2H,PhCH₂), 4.36 (complex m, 1H, H-2), 4.21 (d, J=4.9 Hz, 2H, H-10), 4.08(dd, J=4.8, 1.9 Hz, 1H, H-6), 4.03 (d, J=2.0 Hz, 1H, H-3), 1.32 (d, 3H,J=5.6 Hz, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 137.7 (C, Bn), 134.5 (CH, C-9),130.2 (CH, C-5), 128.48 (CH, Bn), 128.95 (CH, Bn), 127.83 (CH, Bn),125.8 (CH, C-8), 123.3 (C, C-4), 77.7 (CH, C-3), 73.5 (CH, C-7), 72.9(CH, C-6), 71.1 (CH₂, PhCH₂), 67.7 (CH, C-2), 63.1 (CH₂, C-10), 19.5(CH₃, C-1); IR (film from CDCl₃) v_(max) 3628, 3381,3033, 2927, 2630,2339, 1646, 1455, 1345, 1071, 913, 857, 735, 698, 626 cm⁻¹; HRMS: m/zC₁₇H₂₅ ⁸¹BrO₄N⁺[M+NH₄]⁺ calcd 386.0961. found 386.0949.

Synthesis of Compound 18

A sample of diol 17 (45 mg, 0.12 mmol) was dissolved in 1 mL of1,4-dioxane. Then XantPhos (14 mg, 0.0243 mmol, 20%), palladium acetate(6 mg, 0.0243 mmol, 20%), sodium carbonate (65 mg, 0.61 mmol), TBAI (9mg, 0.0243 mmol) and triethylamine (303 μL, 1.2 mmol) were addedsequentially. Then this mixture was bubbled with CO for a few minutes tosaturate the solution with CO. Then the flask was connected to a refluxcondenser and placed under an atmosphere of CO (balloon). The wholeset-up was evacuated and purged with CO three times and stirredvigorously at 95° C. for one day. Then the solution was diluted withdichloromethane (2 mL) and filtered through a silica plug. The crudeproduct was purified by column chromatography (1:1 petroleum ether:ethyl acetate) to yield lactone 18 as a pale yellow oil (20 mg, 52%).18: R_(f) 0.25 (1:1 petroleum ether:ethyl acetate); [α]_(D) ²¹=−60.6 (c0.15, CH₂Cl₂); ¹H-NMR: (CDCl₃) δ_(H) 7.38-7.31 (complex m, 5H, Bn), 7.07(dd, J=7.8, 3.1 Hz, 1H, H-5), 6.09 (dt, J=15.7, 4.7 Hz, 1H, H-9), 6.03(dd, J=15.7, 6.9 Hz, 1H, H-8), 5.03 (dd, J=7.9, 3.3 Hz, 1H, H-3), 4.83(quintet, J=6.8 Hz, 1H, H-2), 4.62 (d, J=11.6 Hz, 2H, PhCH₂), 4.56 (d,J=12.1 Hz, 2H, PhCH₂), 4.37 (dd, J=6.6, 3.8 Hz, 1H, H-7), 4.25 (dd,J=6.2, 4.2 Hz, 1H, H-6), 4.22 (broad d, J=3.8 Hz, 2H, H-10), 1.24 (d,3H, J=6.4 Hz, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 166.2 (C, C-11), 137.3 (C,Bn), 135.7 (CH, C-9), 134.6 (CH, C-5), 131.5 (CH, C-4), 128.57 (CH, Bn),128.17 (CH, Bn), 127.84 (CH, Bn), 125.6 (C, C-8), 77.8 (CH, C-2), 75.3(CH, C-7), 71.6 (CH₂, PhCH₂), 70.4 (CH, C-6), 70.0 (CH, C-3), 62.9 (CH₂,C-10), 15.3 (CH₃, C-1); IR (film from CDCl₃) v_(max) 3628, 3381,3033,2927, 1650, 1413, 1386, 1298, 1096, 829, 736, 689, 632 cm⁻¹; HRMS: m/zC₁₈H₁₈O₅Na⁺[M+Na—H₂O]⁺ calcd 321.1097. found 321.1094.

Synthesis of Compound 19

To a solution of lactone 18 (5.0 mg, 0.016 mmol) in CH₂Cl₂ (0.5 mL) wasadded TiCl₄ (5.2 μL, 0.047 mmol) in CH₂Cl₂ (0.1 mL) at 0° C. Afterstirring at the same temperature for 10 min, the reaction was quenchedwith saturated aqueous NaHCO₃ (2 mL), and the organic layer wasseparated and extracted with CH₂Cl₂ (3×5 mL). The organic layers werecombined and dried over anhydrous MgSO₄. After filtration andconcentration under reduced pressure, the crude product was purified bycolumn chromatography (ethyl acetate) to yield hydroxy-TAN-2483B 19 as acolourless oil (2.6 mg, 72%). 19: R_(f) 0.28 (ethyl acetate); [α]_(D)²¹=−140 (c 0.05, Et₂O); ¹H-NMR: (CDCl₃) δ_(H) 7.09 (dd, J=7.5, 3.5 Hz,1H, H-5), 6.17 (dt, J=15.6, 4.6 Hz, 1H, H-9), 5.98 (dd, J=15.5, 7.0 Hz,1H, H-8), 5.07 (apparent dt, J=6.4 Hz, 1H, H-3), 4.84 (quintet, J=6.8Hz, 1H, H-2), 4.49 (m, 1H, H-6), 4.36 (dd, J=6.8, 3.5 Hz, 1H, H-7), 4.26(broad s, 2H, H-10), 1.27 (d, J=6.6 Hz, 3H, H-1); ¹³C-NMR: (CDCl₃) δ_(C)166.3 (C, C-11), 136.5 (CH, C-9), 135.4 (CH, C-5), 131.3 (C, C-4), 124.3(CH, C-8), 77.8 (CH, C-2), 76.2 (CH, C-7), 70.2 (CH, C-3), 64.2 (CH,C-6), 62.6 (CH₂, C-10), 15.3 (CH₃, C-1); IR (film from Et₂O) v_(max)3701, 3379, 2983, 2870, 2362, 2340, 1755, 1691, 1448, 1358, 1332, 1262,1195, 1141, 1095, 1041, 975, 915, 825, 790, 758, 631, 616 cm⁻¹; HRMS:m/z C₁₁H₁₈O₅N⁺[M+NH₄]⁺ calcd 244.1179. found 244.1186.

Example 4 (−)-TAN-2483B Z-ethyl ester acetate (20) Synthesis of Compound20

To a solution of lactone 10 (3 mg, 0.01 mmol) in CH₂Cl₂ (0.01 mL) wasadded acetic anhydride (10.5 μL, 0.1 mmol) and NEt₃ (8 μL, 0.06 mmol).After stirring room temperature for 5 hours, the crude reaction mixturewas introduced directly to a silica-gel column (2:1 petroleumether:ethyl acetate). The compound 20 was isolated as a colourless oil(1 mg, 32%). 20: R_(f) 0.40 (2:1 petroleum ether:ethyl acetate); ¹H NMR:(CDCl₃) δ_(H) 7.27 (dd, J=6.4, 3.5 Hz, 1H, H-5), 6.32 (dd, J=11.6, 7.4Hz, 1H, H-8), 5.98 (d, J=11.7 Hz, 1H, H-9), 5.50 (dd, J=6.0, 2.2 Hz, 1H,H-6), 5.34 (d, J=7.3 Hz 1H, H-7), 5.12 (dd, J=7.6, 3.5 Hz, 1H, H-3),4.85 (quintet, J=6.8 Hz, 1H, H-2), 4.18 (q, J=7.2 Hz, 2H, OEt), 2.05 (s,3H, CH₃C(O)), 1.30-1.28 (complex m, 6H, OEt and H1); ¹³C NMR: (CDCl₃)δ_(C) 169.9 (C, C-10), 165.3 (C, Acetate), 165.3 (C, C-13), 143.8 (CH,C-8), 132.6 (CH, C-5), 132.5 (C, C-4), 123.0 (CH, C-9), 78.2 (CH, C-2),71.4 (CH, C-3), 70.7 (CH, C-7), 65.2 (CH, C-6), 60.7 (CH₂, OEt), 22.6(CH₃, Acetate), 15.3 (CH₃, OEt or C-1), 14.1 (CH₃, OEt or C-1); HRMS:m/z C₁₃H₂₀NO₆ ⁺[M+NH₄]⁺ calcd 328.1391. found 328.1382.

Example 5 Hydroxy-TAN-2483B diacetate (21) Synthesis of Compound 21

To a solution of diol 19 (3.0 mg, 0.01 mmol) in CH₂Cl₂ (0.1 mL) wasadded acetic anhydride (16 μL, 0.1 mmol) and triethylamine (21 μL, 0.2mmol). After stirring at room temperature for two hours, the crudeproduct was purified by column chromatography (gradient column, 9:1,5:1, 2:1, 1:1 petroleum ether:ethyl acetate) to yield 21 as a colourlessoil (1.5 mg, 36%). 21: R_(f) 0.54 (1:1 petroleum ether:ethyl acetate);[α]_(D) ²⁵=−320 (c 0.075, Et₂O); (CDCl₃) δ_(H) 7.03 (dd, J=4.8, 3.2 Hz,1H, H-5), 6.01 (dt, J=15.6, 5.7 Hz, 1H, H-9), 5.91 (dd, J=15.6, 7.1 Hz,1H, H-8), 5.42 (apparent dt, J=6.4 Hz, 1H, H-6), 5.02 (dq, J=7.8 Hz,3.3, 1H, H-3), 4.85 (quintet, J=6.8 Hz, 1H, H-2), 4.61 (d, J=5.4 Hz, 2H,H-10), 4.51 (dd, J=7.1, 3.9, 1H, H-7), 2.09 (s, 3H, CH₃C(O)), 2.08 (s,3H, CH₃C(O)), 1.27 (d, J=6.3 Hz, 3H, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 170.5(C, Acetate), 170.1 (C, Acetate), 165.7 (C, C-11), 132.8 (C, C-4), 131.9(CH, C-5), 131.1 (CH, C-9), 127.1 (CH, C-8), 77.8 (CH, C-2), 73.9 (CH,C-7), 70.0 (CH, C-3), 65.6 (CH, C-6), 63.7 (CH₂, C-10), 20.9 (CH₃,Acetate), 20.6 (CH₃, Acetate), 15.3 (CH₃, C-1); HRMS: m/zC₁₅H₂₂O₇N⁺[M+NH₄]⁺ calcd 328.1391. found 328.1402.

Example 6 Methoxy-TAN-2483B (22) Synthesis of Compound 22

To a solution of proton sponge (33.90 mg, 0.154 mmol) in CH₂Cl₂ (0.3 mL)was added Meerwein salt (17.6 mg, 0.11 mmol). After cooling thesuspension to 0° C., diol 19 (3 mg, 0.01 mmol) in CH₂Cl₂ (0.3 mL) wasadded. After stirring at the same temperature for one hour, the reactionwas slowly warmed to room temperature and stirred for four hours. Thecrude reaction mixture was directly subjected to column chromatography(gradient column, 9:1, 5:1, 2:1, 1:1 petroleum ether:ethyl acetate) toyield 22 as a colourless oil (1.2 mg, 38%). 22: R_(f) 0.13 (1:1petroleum ether:ethyl acetate); [α]_(D) ²⁵=−78.8 (c 0.06, Et₂O); ¹H-NMR:(CDCl₃) δ_(H) 7.09 (dd, J=4.7, 3.6 Hz, 1H, H-5), 6.08 (dt, J=15.6, 5.2Hz, 1H, H-9), 5.94 (dd, J=15.6, 7.1 Hz, 1H, H-8), 5.07 (apparent m, 1H,H-3), 4.84 (quintet, =6.9 Hz, 1H, H-2), 4.49 (m, 1H, H-6), 4.48 (dd,J=6.8, 3.5 Hz, 1H, H-7), 3.99 (dd, J=1.5, 0.8 Hz, 2H, H-10), 3.38 (s,3H, OCH₃), 1.27 (d, J=6.3 Hz, 3H, H-1); ¹³C-NMR: (CDCl₃) δ_(C) 169.3 (C,C-11), 135.4 (CH, C-5), 135.1 (CH, C-9), 131.3 (C, C-4), 125.5 (CH,C-8), 77.8 (CH, C-2), 76.2 (CH, C-7), 72.0 (CH₂, C-10), 70.2 (CH, C-3),64.2 (CH, C-6), 58.44 (CH₃, OMe), 15.3 (CH₃, C-1); IR (film from Et₂O)v_(max) 2975, 2861, 1444, 1381, 1350, 1297, 1117, 1075, 1044, 934, 880,844, 794, 499, 440 cm⁻¹; HRMS: m/z C₂₄H₃₂O₁₀N⁺[2M+NH₄]⁺ calcd 499.2367.found 499.2381.

Example 7 Cancer Cell Growth Inhibition

HL-60, 1A9 and MCF cells were cultured at 37° C. in a 5% CO₂ in airatmosphere in RPMI-1640 medium supplemented with 10% fetal calf serum,100 units/mL penicillin, and 100 units/mL streptomycin (with 0.1%insulin added to the medium for 1A9 and MCF7 cell cultures). An MU cellproliferation assay was used that involved the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoyltetrazolium bromide (MU) byviable cells as previously described.¹⁴ Cells were treated withcompounds of the invention in 96-well plates (duplicate wells) for 2days (for HL-60) or 3 days (for 1A9 or MCF7), then incubated with 5mg/mL MU in phosphate-buffered saline for 2 h. The blue crystals thatformed were solubilised in 10% sodium dodecyl sulfate, 45%dimethylformamide and the absorbance of the solutions measured at 570 nmin a multiwell plate reader (EnSpire™ 2300 Multiplate Reader from PerkinElmer, Waltham, USA. The half-maximal inhibitory concentration (IC₅₀)was calculated from a concentration-response curve using Sigma Plotsoftware v8 (Systat Software Inc. Point Richmond, Calif.).

TABLE 2 Cancer cell line inhibition results Compound 10 Compound 14Compound 19 Cell Line (IC₅₀ μM) (IC₅₀ μM) (IC₅₀ μM) HL-60 3.6 2.2 42MCF7 9.0 — — 1A9 — 3.4 —

Example 8 Kinase Inhibition

Compound 10 was assessed for kinase inhibition by Life Technologiesusing the SelectScreen® Whole Panel ACCESS Biochemical Kinase ProfilingService. The Z'-LYTE® Screening Protocol was used to obtain the resultsfor the following kinases: AMPK A2/B1/G1, BMX, BTK, MAPK14 (p38 alpha),PLK1, TXK. The LanthaScreen Protocol was used to obtain the result forthe following kinase: NUAK2.

TABLE 2 Kinase inhibition results for compound 10 Inhibition Kinase(IC₅₀ at 10 μM) AMPK A2/B1/G1 81 BTK 83 PLK1 81 BMX 80 NUAK2 76 MAPK1474 TXK1 71

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention as defined in the claims.Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically referred in thisspecification.

LIST OF REFERENCES

-   1. Krohn, K., et al., Eur. J. Org. Chem., 2002, 2331-2336.-   2. Qin, S., et al., Eur. J. Org. Chem., 2009, 3279-3284.-   3. Oh, H., et al., Tetrahedron Lett., 2001, 42, 975-977.-   4. Kock, I., et al., Eur. J. Org. Chem., 2007, 2186-2190.-   5. Krohn, K., et al., Chirality, 2007, 19, 464-470.-   6. Japanese Patent 10287679, 1998: Chem. Abstr., 1999, 130, 3122e.-   7. Nozawa, O., et al., J. Antibiot., 1995, 48, 113-118.-   8. Nozawa, O., et al., J. Antibiot., 2000, 53, 1296-1300.-   9. Gao, X., et al., Org. Lett., 2003, 5, 451-454.-   10. Gao, X. and Snider, B. B., J. Org. Chem., 2004, 69, 5517-5527.-   11. Shaabani, A., et al., Bioorg. Med. Chem. Lett., 2008, 18,    3968-3970.-   12. Hewitt, R. J., and Harvey, J. E., Org. Biomol. Chem., 2011, 9,    998-1000.-   13. Kim, C., et al., Org. Lett., 1999, 1, 1295-1297.-   14. Hood, K. A., et al., Apoptosis, 2001, 6, 207-219.

1. A compound of the formula:

wherein R¹ and R² may each be H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆alkenyl, C₂-C₆ alkynyl, aryl, CO₂H, CO₂alkyl, or C(═O)alkyl, whereineach alkyl, alkoxy, alkenyl, alkynyl or aryl group may optionally besubstituted with OH, NH₂, halogen, alkoxy, acyloxy or aryl; and R³ is H,C₁-C₆ alkyl, C₁-C₆ acyl, aryl, benzyl or trialkylsilyl; provided that R¹is not CH₃ when R² and R³ are both H or when R² is H and R³ is acetyl;or a pharmaceutically acceptable salt thereof.
 2. A compound as claimedin claim 1, wherein: R¹ and R² may each be H, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, CO₂H, CO₂alkyl, or C(═O)alkyl,wherein each alkyl, alkoxy, alkenyl, alkynyl or aryl group mayoptionally be substituted with OH, NH₂, halogen, or aryl; and R³ is H,C₁-C₆ alkyl, aryl, benzyl or trialkylsilyl; provided that R¹ is not CH₃when R² and R³ are both H or when R² is H and R³ is acetyl.
 3. Acompound as claimed in claim 1, wherein one of R¹ and R² is C₁-C₆ alkyland the other is H.
 4. A compound as claimed in claim 1, wherein one ofR¹ and R² is CH₂OH and the other is H.
 5. A compound as claimed in claim1, wherein one of R¹ and R² is CO₂alkyl and the other is H.
 6. Acompound as claimed in claim 1, wherein one of R¹ and R² is CO₂H and theother is H.
 7. A compound as claimed in claim 1, wherein R³ is H.
 8. Acompound as claimed in claim 1, wherein R³ is methyl, ethyl or acetyl.9. A compound selected from the group consisting of:


10. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 11. A method of treating cancer,osteoporosis, Type 2 diabetes, or an immune disease comprisingadministering a pharmaceutically effective amount of a compound of claim1 to a patient requiring treatment.
 12. A method as claimed in claim 11,wherein the cancer is leukaemia, ovarian cancer or breast cancer.
 13. Amethod as claimed in claim 11, wherein the immune disease is asthma,eczema, allergic rhinitis, Type 1 diabetes, rheumatoid arthritis orlupus.
 14. A compound as claimed in claim 2, wherein one of R¹ and R² isC₁-C₆ alkyl and the other is H.
 15. A compound as claimed in claim 2,wherein one of R¹ and R² is CH₂OH and the other is H.
 16. A compound asclaimed in claim 2, wherein one of R¹ and R² is CO₂alkyl and the otheris H.
 17. A compound as claimed in claim 2, wherein one of R¹ and R² isCO₂H and the other is H.
 18. A compound as claimed in claim 3, whereinR³ is H.
 19. A compound as claimed in claim 3, wherein R³ is methyl,ethyl or acetyl.
 20. A compound as claimed in claim 4, wherein R³ is H.